This invention relates to immunoassays, and more particularly to such assays wherein a fluorescent tag capable of emitting fluorescent radiation when excited by more energetic exciting radiation is incorporated into a constituent of an antigen-antibody or similar complex.
Immunoassays, in which aliquots of sample and one or more reagents are variously reacted to form antigen-antibody or similar complexes which may then be observed in order to assay the sample for the presence and titer of a predetermined moiety of the complex, are well known. Typical of such assays are those wherein a specific antibody is used to measure the quantity of the antigen for which it is specific (or vice versa). However, the technique has been extended to quantitate haptens (including hormones, alkaloids, steroids, and the like) as well as antigens, and antibody fragments (i.e., Fab) as well as complete antibodies, and it is in this broader sense that the present invention should be understood.
As is well known, sensitive immunoassays employ tracer techniques wherein a tagged constituent of the complex is incorporated into the reagent, the non-complexed tagged reagent being separated from the complexed reagent, and the complex (or non-complexed reagent) then quantitated by observing the tag. Both radioisotopes and fluorescent markers have been used to tag constituents of immunoassay reagents, the tag being respectively observed by a gamma ray counter or a fluorometer. The present invention is, however, directed only to those assay which rely on fluorescence.
The separation of the non-complexed tagged moiety from the complexed is commonly accomplished by immobilizing a predetermined one of the components of the complex to a solid phase (such as the inside wall of a test tube, glass or polymeric beads, or the like) in such a way as not to hinder the component's reactivity in forming the complex. As an example, an antibody such as immunoglobulin G (IgG) may be bound by its carboxyl terminations to a solid phase, such as glass, by a silyl compound such as 3-aminopropyltrimethoxysilane, thereby leaving the antibody's antigen reactive amino terminations free. Any complex formed incorporating the immobilized component may then be physically separated from the non-reacted complement remaining in solution, as by aspirating or decanting the fluid from a tube or eluting the fluid through a particulate bed.
In competition immunoassay, the reagent consists of a known quantity of tagged complement (such as antigen) to the immobilized component of the complex (in this instance, antibody). The reagent is mixed with a fixed quantity of the sample containing the untagged complement to be quantitated. Both tagged and untagged complement attach to the immobilized component of the complex in proportion to their relative concentrations. After incubation for a set time, the fluid sample and reagent are separated. The complex immobilized to the solid phase is then illuminated with radiation of a wavelength chosen to excite fluorescense of the tag, and the fluorescense is measured. The intensity of the fluorescense of the immobilized complex is inversely proportional to the concentration of the untagged complement being assayed.
Alternatively, an assay may be made by immobilizing a quantity of an analog of the moiety to be quantitated (i.e., a substance which is immunologically similarly reactive) and reacting the sample with a known quantity of tagged complement. The tagged complement complexes with both the unknown quantity of the moiety in the sample and the immobilized analog. Again, the intensity of fluorescence of the immobilized complex is inversely proportional to the concentration of the (free) moiety being quantitated.
So-called "sandwich" immunoassays may be performed for multivalent complements to the immobilized component, the attached complement being then further reacted with a tagged analog of the imobilized component. Thus, bivalent antigen may be bound to an immobilized antibody and then reacted with a fluorescent tagged antibody, forming an antibody--antigen--tagged antibody sandwich that may then be separated from the unreacted tagged antibody. The intensity of the fluorescence of the thus formed immobilized complex is directly proportional to the concentration of the species being quantitated.
In any of the assays, accuracy in quantitation is determined in part by the technique of the laboratory personnel performing the assay. Thus, precision fluorescence immunoassay requires fluorometric measurement of a predetermined volume of the sample to which a predetermined quantity of reagent has been added at a known dilution. To insure that the necessary volume measurements are not the accuracy limiting step of the assay requires that the assay be performed by skilled personnel and often with precision apparatus, or alternatively, precisely constructed and preloaded disposable reagent kits (to insure the titer of reagent) together with an accurately timed diffusion process (to insure the size of the sample volume assayed.
It will be appreciated that the use of skilled personnel, precision apparatus, and accurate manipulative or timing requirements impact both the cost and wide-scale availability of an assay.